吡格列酮对高糖诱导的大鼠肾小管上皮细胞-间充质细胞转分化的影响及机制研究
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篇名: 吡格列酮对高糖诱导的大鼠肾小管上皮细胞-间充质细胞转分化的影响及机制研究
TITLE: Effects and Mechanism Study of Pioglitazone on High Glucose-induced Epithelial-mesenchymal Transition in Renal Tubular Epithelial Cells of Rat
摘要: 目的:探讨吡格列酮(PIO)对高糖诱导的大鼠肾小管上皮细胞-间充质细胞转分化(EMT)的影响及可能机制,为防治糖尿病肾病提供理论依据及新靶点。方法:将大鼠肾小管细胞NRK-52E随机分为对照组(5.5mmol/L葡萄糖)、高糖组(30mmol/L葡萄糖)、PIO干预组(30mmol/L葡萄糖+5.0μmol/LPIO)、GW9662干预组(30mmol/L葡萄糖+5.0μmol/LPIO+5.0μmol/L特异性拮抗剂GW9662)。前3组细胞分别在培养6、12、24、48h时进行动态检测,GW9662干预组细胞在培养48h时进行检测。采用Real-timePCR法检测细胞中第10号染色体缺失的磷酸酶和张力蛋白同源基因(PTEN)、过氧化物酶体增殖物激活受体γ(PPARγ)mRNA的表达水平;采用Westernblotting法检测细胞中PTEN、PPARγ、α-平滑肌肌动蛋白(α-SMA)、上皮钙黏素(E-cadherin)的表达以及磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(AKT)信号通路的变化。结果:随培养时间的延长,与正常组比较,高糖组细胞中PPARγ、PTENmRNA及蛋白(除PPARγ6h外)表达水平均显著降低,α-SMA、p-AKT(Thr308)蛋白表达水平均显著升高,E-cadherin蛋白表达水平显著降低(P<0.05),且呈时间依赖趋势;与高糖组比较,PIO组细胞中PPARγ(除6h时的蛋白表达外)、PTEN的mRNA及蛋白表达水平均显著升高,α-SMA、p-AKT(Thr308()除6h外)蛋白表达水平均显著降低,E-cadherin蛋白表达水平显著增高(P<0.05),且呈时间依赖趋势。与高糖组比较,GW9662干预组细胞PTEN、PPARγmRNA及蛋白表达水平,α-SMA、E-cadherin、p-AKT(Thr308)蛋白表达水平的差异均无统计学意义,PIO的作用效应被PPARγ拮抗剂GW9662阻断。结论:在高糖条件下PIO可能是通过激活PPARγ而实现对PTEN表达的调控,使PI3K/AKT信号通路受到抑制,进而抑制肾小管上皮细胞EMT的发生。
ABSTRACT: OBJECTIVE:To investigate the effects o f pioglitazone (PIO)on high glucose-induced epithelial-mesenchymal transition(EMT)in renal tubular epithelial cells of rat and its possible mechanism ,and to provide theoretic reference and new target for the prevention and treatment of diabetic nephropathy. METHODS :The rat renal tubular epithelial NRK- 52E cells were randomly divided into control group (5.5 mmol/L glucose ),high-glucose group (30 mmol/L glucose ),PIO intervention group (30 mmol/L glucose+ 5.0 μmol/L PIO),GW9662 intervention group (30 mmol/L glucose+ 5.0 μmol/L PIO+5.0 μmol/L specific anta- gonist GW 9662). The cells of the first 3 groups were detected at 6,12,24,48 h of culture ,while those in GW 9662 intervention group were detected at 48 h of culture. mRNA expression of PTEN and PPARγ were detected by real-time PCR. The protein expression of PTEN ,PPARγ,α-SMA and E-cadherin as well as the changes of PI 3K/AKT signaling pathway were determined by Western blotting assay. RESULTS :With the extension of culture time ,compared with control group ,the mRNA and protein expression of PPARγ(except for protein expression at 6 h)and PTEN in high-glucose group reduced significantly ,while the protein expression of α-SMA and p-AKT (Thr308)increased significantly ,and the protein expression of E-cadherin reduced significantly (P<0.05),showing time-dependent trend. Compared with high-glucose group ,the mRNA and the protein expression (except for 6 h) of PPAR γ and PTEN were increased significantly in PIO intervention group , while the protein expression of α-SMA and p-AKT (Thr308) were decreased significantly,and the protein expression of E-cadherin wasincreased significantly (P<0.05), showing time-dependent trend. There was no statistical significance in mRNA and protein expression of PPAR γ and PTEN,protein expression of E-cadherin ,α-SMA and p-AKT (Thr308) between GW 9662 intervention group and high-glucose group ;the effect of PIO was blocked by PPAR γ antagonist GW9662. CONCLUSIONS :PIO may up-regulate the expression of PTEN by activating PPARγ,inhibit PI 3K/AKT signaling pathway so as to inhibit the occurrence of EMT of renal tubular epithelial cells .
期刊: 2020年第31卷第16期
作者: 孙兰,张帆,石明隽,田平平,郭兵
AUTHORS: SUN Lan,ZHANG Fan,SHI Mingjun ,TIAN Pingping ,GUO Bing
关键字: 吡格列酮;肾小管上皮细胞;过氧化物酶体增殖物激活受体γ;第10号染色体缺失的磷酸酶和张力蛋白同源基因;磷脂酰
KEYWORDS: Pioglitazone;Renal tubular epithelial cell ;PPARγ;PTEN;PI3K/AKT signaling pathway ;Epithelial-mesenchymal
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